Role of TREM2 gain-of-function mutations in modulating microglial pathology in Alzheimer's disease

项目摘要 全基因组关联研究已经确定了许多与免疫相关的阿尔茨海默病（AD）风险基因。 反应其中包括小胶质细胞受体CD 33和TREM 2。我们之前展示了敲除 在5XFAD小鼠中，CD 33的表达减弱了淀粉样β蛋白（Aβ）的病理学并改善了认知能力，两者都是 通过额外的Trem 2敲除而消除。在5XFAD小鼠中敲除Trem 2会加重Aβ病理学， 神经变性，但减少小胶质细胞数量。小胶质细胞的RNA-seq分析显示， 在5XFAD; Trem 2-/-小鼠中，对吞噬作用和信号传导（IL-6、IL-8、急性期反应）的调节下调。 5XFAD中的差异基因表达; CD 33-/-小胶质细胞依赖于Trem 2的存在，表明 TREM 2作用于CD 33的下游。此外，我们报道了TREM 2的D87 N和T96 K变体， 在NIMH家族遗传数据和ADSP病例对照样本中，AD风险增加， 这些变体增加了与TREM 2配体的结合。在我们的初步研究中，我们发现， Trem 2 T96 K变体增加了不溶性Aβ42的水平，但减少了小胶质细胞的数量和聚集。 5XFAD小鼠中Aβ斑块周围的小胶质细胞。此外，Trem 2 T96 K变体减少了sTrem 2的分泌， 5XFAD小鼠。最后，T96 K变体导致细胞表面TREM 2的表达和分泌减少。 人小胶质细胞系。在这里，我们建议1）评估TREM 2功能获得性突变对 AD发病机制，2）研究这些突变对小胶质细胞活化和基因表达的影响， 和3）鉴定和表征新的AD相关TREM 2功能获得性突变以及靶向 TREM 2用于治疗目的。在目标1中，我们将检查功能增益Trem 2 T96 K的影响 突变对5XFAD小鼠中Aβ病理学、神经变性和斑块相关神经炎性营养不良的影响。我们 还将确定Trem 2 T96 K是否影响5XFAD小鼠的认知功能。在目标2中，我们将研究 功能获得性Trem 2 T96 K突变对小胶质细胞活化和募集Aβ的影响 斑块以及对5XFAD小鼠中脂多糖刺激的免疫应答。RNA-seq分析 小胶质细胞将揭示Trem 2 T96 K对小胶质细胞基因表达特征的影响，以及它们与 疾病相关的小胶质细胞在目标3中，我们将分析来自NIMH家族的全基因组测序数据 样本和NIA ADSP，以鉴定新的AD相关TREM 2突变，这些突变将被表征为它们的 对TREM 2的配体依赖性活化的影响。然后，我们将介绍新型TREM 2功能增益 使用CRISPR/Cas9系统将突变导入iPSC衍生的人小胶质细胞样细胞中，并研究其潜在的生物学效应。 分子机制最后，我们将预测和验证模拟TREM 2激活的新型靶向药物。 和/或使用计算方法对抗TREM 2功能丧失。这项建议的主要目标是 全面评估TREM 2功能获得性突变对AD发病机制的影响，并探索 潜在的分子网络，这将有助于AD治疗靶向TREM 2。

Project Summary Genome-wide association studies have identified many Alzheimer’s disease (AD) risk genes related to immune response. Among these are the microglial receptors CD33 and TREM2. We previously showed that knock-out of CD33 attenuated amyloid beta (Aβ) pathology and improved cognition in 5XFAD mice, both of which were abrogated by additional Trem2 knock-out. Knocking out Trem2 in 5XFAD mice exacerbated Aβ pathology and neurodegeneration but reduced microglia numbers. RNA-seq profiling of microglia revealed that genes related to phagocytosis and signaling (IL-6, IL-8, acute phase response) are downregulated in 5XFAD;Trem2-/- mice. Differential gene expression in 5XFAD;CD33-/- microglia depended on the presence of Trem2, suggesting TREM2 acts downstream of CD33. Moreover, we reported that the D87N and T96K variants of TREM2 increased AD risk in the NIMH family-based genetic data and ADSP case-control samples and showed that these variants increased binding to TREM2 ligands. In our preliminary study, we found that the gain-of-function Trem2T96K variant increased levels of insoluble Aβ42 but reduced both microglia numbers and clustering of microglia around Aβ plaques in 5XFAD mice. Furthermore, the Trem2T96K variant reduced secretion of sTrem2 in 5XFAD mice. Finally, the T96K variant led to reduced cell surface expression and secretion of TREM2 in human microglial cell lines. Here, we propose to 1) assess the impact of TREM2 gain-of-function mutations on AD pathogenesis, 2) investigate the effects of these mutations on microglial activation and gene expression, and 3) identify and characterize novel AD-associated TREM2 gain-of-function mutations as well as target TREM2 for therapeutic purposes. In Aim 1, we will examine the effects of the gain-of-function Trem2T96K mutation on Aβ pathology, neurodegeneration and plaque associated-neuritic dystrophy in 5XFAD mice. We will also determine whether Trem2T96K impacts cognitive function in 5XFAD mice. In Aim 2, we will investigate the impact of the gain-of-function Trem2T96K mutation on microglial cell activation and recruitment to Aβ plaques as well as immune response to lipopolysaccharide stimulation in 5XFAD mice. RNA-seq profiling of microglia will reveal the effects of Trem2T96K on microglial gene expression signature and how they compare to disease-associated microglia. In Aim 3, we will analyze whole genome sequencing data from the NIMH family sample and NIA ADSP to identify novel AD-associated TREM2 mutations that will be characterized for their effects on ligand-dependent activation of TREM2. We will then introduce novel TREM2 gain-of-function mutations into iPSC-derived human microglia-like cells using CRISPR/Cas9 system and investigate underlying molecular mechanisms. Finally, we will predict and validate novel targeted drugs that mimic TREM2 activation and/or oppose TREM2 loss-of-function using computational approaches. The major goals of this proposal are to comprehensively assess the effects of TREM2 gain-of-function mutations on AD pathogenesis and explore underlying molecular networks, which will facilitate AD therapeutics targeting TREM2.